Osel, Inc. is a biopharmaceutical company specializing in the development of bacterial therapeutics. The company's lead product, LACTIN-V, is a naturally occurring human vaginal isolate of Lactobacillus crispatus presently undergoing phase II clinical trials to examine its safety and efficacy in preventing recurrent urinary tract infections (UTIs) and bacterial vaginosis (BV). Both UTIs and BV are associated with a depletion of hydrogen peroxide (H2O2)-producing lactobacilli that normally protect the vagina from infection by opportunistic pathogens. Epidemiological studies also suggest that loss of vaginal lactobacilli is associated with an increased risk of heterosexual HIV-1 transmission and other sexually transmitted infections. LACTIN-V represents an ecological approach to prevent vaginal infections by re-establishing the protective vaginal flora with a colonizing, H2O2-producing Lactobacillus strain. A second-generation Lactobacillus product, currently under development, is a human vaginal isolate of H2O2-producing L. jensenii that has been genetically modified to constitutively secrete high levels of a single protein-based potent HIV envelope-targeted entry inhibitor, an N-terminally modified cyanovirin-N (CV-N) (P51G). This live, self-renewing microbicide may afford an efficacious, yet inexpensive means to deliver a protein-based microbicide and address the urgent need for female-controlled approaches to block heterosexual transmission of HIV-1. Among the microbicide candidates currently in clinical trials, almost all of them are "coital dependent" products, or are being formulated for development based on a single active ingredient. The need to develop "non-coital dependent" microbicides or combinational microbicides that act at multiple sites of the mucosal HIV infection pathway represents a significant gap in microbicide development. In this R21 proposal, we plan to develop a novel live combinational microbicide by employing an H2O2-producing vaginal L. jensenii 1153 genetically modified to constitutively secrete the N-terminally modified CV-N (P51G) and an effective CCR5- targeted entry inhibitor, C1C5-RANTES. The latter is an N-terminally modified RANTES that targets the major HIV co-receptor CCR5 with an increased antiviral and anti-inflammatory activity. In this proposal, we will explore the possibility of delivering CV-N and C1C5-RANTES from a single strain of Lactobacillus, or from a mixture of two strains, each delivering a single inhibitor. We will select a microbicide development candidate from a collection of bioengineered strains that contain optimized expression cassettes stably integrated into the L. jensenii chromosome. The R33 phase will follow after a successful completion of the proposed milestones in the R21 phase proposal. For the R33 phase, we propose to conduct preclinical animal safety and efficacy studies in mouse, rabbit, and non-human primates. We will employ a Chinese rhesus macaque (Macaca mulatta) model that allows persistent vaginal colonization of L. jensenii to conduct preclinical safety and efficacy studies, including in situ CV-N and C1C5-RANTES expression, immunotoxicity, and efficacy against mucosal viral transmission. Furthermore, we propose to evaluate potential regulatory issues concerning the pharmaceutical development of a genetically modified organism which delivers combinational microbicides and to have a pre-IND consultation with FDA.